The major barrier to the use of stem cell therapy in regenerative medicine is the inability to regulate the dichotomous capacity for stem cell self-renewal versus the process of cell lineage commitment. The solution to this problem will require an improved understanding of the inductive signals and the cognate signal transduction pathways which determine cellular fate, and which specifically govern the competitive outcomes of self-renewal with maintenance of pluripotency, versus differentiation into a specialized tissue phenotypei.
The evolutionarily conserved canonical Wnt pathway has been implicated in both human and mouse embryonic stem (ES) cell self-renewal competenceii. Inactivation of glycogen synthase kinase-3β (GSK-3β) leads to nuclear accumulation of β-catenin, which, in turn, leads to the activation of Wnt target genes implicated in the proliferation of endothelial precursor cellsiii, and in self-renewal of HESCsiv.
ILK is a protein Ser/Thr kinase that binds to the cytoplasmic domains of β1, β2 and β3-integrin subunitsv. ILK is regulated in a phosphoinositide 3′-kinase (PI3K)-dependent manner following distinct signal inputs from integrins and growth factor receptor tyrosine kinasesvi,vii. Conditional knockdown and RNA interference experiments indicate that ILK is required for phosphorylation of PKB/Akt Ser473 and GSK-3β Ser9viii. Since inhibitory phosphorylation of GSK-3β is sufficient for maintenance of an undifferentiated phenotype in mouse and human ESCs, ILK is a candidate kinase activator of a critical stem cell signaling cascade.
We have shown that cardiac-restricted ILK over-expression in a mouse model causes a compensatory (beneficial) form of cardiac hypertrophy. Molecular analysis revealed that ILK mediated hypertrophy is dependent upon a novel pathway involving activation of the small G-protein, Rac1. Gene expression profiling of ILK transgenic mice subjected to LAD ligation-induced myocardial infarction revealed up-regulation of transcripts linked to IL-6 and Janus-associated tyrosine kinase/signal transducer of activated transcription (JAK/STAT3) signaling. These studies establish ILK as an important new cardiovascular target. The activation of these signaling cascades in this myocardial injury model should be stimulative to stem cell recruitment based on their established role in cell renewal in mouse ESCs.
We anticipate that fetal sources of tissue will be enriched for stem cells, given that stem cell activation recapitulates fetal programming. We have developed and characterized an in vitro model of human fetal cardiac myocytes (HFCM)ix, and characterized the genomic response to ischemic stress during human heart surgery in vivox. We have shown that cardiac stem-like cells can be identified by c-kit staining in HFCM with a frequency approximately one order of magnitude higher than that described for adult heartxi. Further, we have shown that ILK gain-of-function increases the frequency of c-kit- and CD133-positive cardiac progenitor cells isolated from human myocardium, highlighting this as a rational approach to augment stem cell-based cellular therapy.
Ventricular hypertrophy is an extremely common clinical condition that appears as a consequence of any variety of volume and or pressure overload stresses on the human heart. An increase in ventricular mass occurring in response to increased cardiac loading is generally viewed as a compensatory response, which serves to normalize ventricular wall tension and improve pump function. Conversely, a sustained or excessive hypertrophic response is typically considered maladaptive, based on the progression to dilated cardiac failure sometimes observed clinically, and the statistical association of ventricular hypertrophy with increased cardiac mortality. Whereas mouse models of cardiac hypertrophy have been generated by genetically-induced alterations in the activation state of various kinases in the heart, limited information is available regarding the role of specific signaling pathways activated during human ventricular hypertrophy.
The identification of the kinase pathways implicated in human hypertrophy has important therapeutic implications, since it will allow testing of the hypothesis that enforced hypertrophy induction represents a beneficial remodeling response, and a useful strategy to preserve cardiac function and arrest the transition to a dilated phenotype.